Selected nucleotide sequences isolated from pathogenic strains of haemophilus influenzae

ABSTRACT

A DNA sequence of  Haemophilus influenzae  clone 151_04 shown in SEQ. ID. NO. 1. A DNA sequence of  Haemophilus influenzae  clone 125_L2 shown in SEQ. ID. NO. 2. A DNA sequence of  Haemophilus influenzae  clone 179_D14 shown in SEQ. ID. NO. 3. A DNA sequence of  Haemophilus influenzae  clone 167_A16 shown in SEQ. ID. NO. 4.

This application is a continuation of application Ser. No. 10/698,235filed on Oct. 31, 2003 now U.S. Pat. No. 6,969,762.

FIELD OF THE INVENTION

The present invention is related to Haemophilus influenzae. Morespecifically, the present invention is related to selected nucleotidesequences isolated from pathogenic strains of Haemophilus influenzae.

BACKGROUND OF THE INVENTION

Haemophilus influenzae is a naturally transformable gram-negativebacterial pathogen that colonizes both the upper and lower humanrespiratory tracts. In the upper respiratory tract it can colonize thenasopharynx and then spread locally to cause disease in the ears (otitismedia), sinuses (sinusitis), and meninges (meningitis). In the lowerrespiratory tract H. influenzae is associated with chronic obstructivepulmonary disease (COPD), bronchitis, and pneumonia. H. influenzae issometimes considered to be an opportunistic pathogen as invasion usuallyfollows a loss of the mucocilliary escalator function following eitherviral or toxigenic denudement. (Swords, W. E., M. R. Ketterer, J. Shao,C. A. Campbell, J. N. Weiser, and M. A. Apicella. 2001. Binding of thenon-typeable Haemophilus influenzae lipooligosaccharide to the PAFreceptor initiates host cell signalling. Cell. Microbiol. 3:525–536,incorporated by reference herein.) H. influenzae is associated with bothacute and chronic infections for which it has developed specializedsurvival strategies.

The entire genome of the less virulent laboratory reference strain of H.influenzae, designated Rd, was sequenced and published in1995-(Fleischmann et al., Science 269:496–512). However, since many ofthe genes that are responsible for the virulence of H. influenzae aremissing from strain Rd, the sequence of the Rd genome is insufficientfor developing tools to detect or prevent H. influenzae infections.

H. influenzae strains are classified according to their expression, orlack of expression, of a polysaccharide capsule. Encapsulated isolatesare divided into six antigenic serotypes (a–f), whereas nonencapsulatedisolates are classified as nontypeable H. influenzae (NTHi). H.influenzae type b (Hib) is associated with invasive disease and waspreviously responsible for the vast majority of H. influenzae-associatedcases of meningitis. The introduction of an Hib vaccine in 1985 greatlydecreased the incidence of type b infections (Scheifele, D. W., T. P.Jadavji, B. J. Law, R. Gold, N. E. Macdonald, M. H. Lebel, E. L. Mills,P. Dery, S. A. Halperin, R. F. Morris, V. Marchessault, and P. J.Duclos. 1996. Recent trends in pediatric Haemophilus influenzae type binfections in Canada. Can. Med. Assoc. J. 154:1041–1047; Schulte, E. E.,G. S. Birkhead, S. F. Kondracki, and D. L. Morse. 1994. Patterns ofHaemophilus influenzae type b invasive disease in New York State,1987–1991: the role of vaccination requirements for day-care attendance.Pediatrics 94:1014–1016, both of which are incorporated by referenceherein); however, non-type b strains, including the NTHi, continue to beimportant pathogens worldwide.

Most strains of H. influenzae are naturally competent, with the abilityto take up DNA from their environments and integrate it into theirchromosomes. Many naturally competent bacteria such as Haemophilus sp.and Neisseria sp. preferentially take up DNA from highly relatedorganisms, which they recognize by the presence of genera-specificconserved uptake signal sequences (USSs) that are over-represented intheir respective genomes (Elkins, C., C. E. Thomas, H. S. Seifert, andP. F. Sparling. 1991. Species-specific uptake of DNA by gonococci ismediated by a 10-base-pair sequence. J. Bacteriol. 173:3911–3913;Mathis, L. S., and J. J. Scocca. 1982. Haemophilus influenzae andNeisseria gonorrhoeae recognize different specificity determinants inthe DNA uptake step of genetic transformation. J. Gen. Microbiol.128:1159–1161; Smith, H. O., J.-F. Tomb, B. A. Dougherty, R. D.Fleischmann, and J. C. Venter. 1995. Frequency and distribution of DNAuptake signal sequences in the Haemophilus influenze Rd genome. Science269:538–540, all of which are incorporated by reference herein). The H.influenzae Rd genome contains 1465 copies of the hUSS (Smith, H. O.,J.-F. Tomb, B. A. Dougherty, R. D. Fleischmann, and J. C. Venter. 1995.Frequency and distribution of DNA uptake signal sequences in theHaemophilus influenze Rd genome. Science. 269:538–540, incorporated byreference herein); thus, on average, there is about one hUSS per 1200bases, or approximately one/gene. This frequency of the hUSS wouldtheoretically provide for the exchange of any H. influenzae gene amongstrains. Thus, the natural transformation system of H. influenzaeprovides a mechanism for generating multiple types of genetic diversityamong strains, including genetic heterogeneity (allelic differences) andgenomic plasticity (genic differences).

There is evidence that inter-species transfers occur as well. Kroll etal. (Kroll, J. S., K. E. Wilks, J. L. Farrant, and P. R. Langford. 1998.Natural genetic exchange between Haemophilus and Neisseria: intergenerictransfer of chromosomal genes between major human pathogens. Proc. Natl.Acad. Sci. USA 95:12381–12385, incorporated by reference herein) havepresented evidence of uptake sequence-mediated intergeneric transferbetween the upper respiratory pathogens Haemophilus and Neisseria. Theseresearchers identified three regions of Haemophilus-like DNA in theNeissera meningitidis genome and suggested that transformation withheterologous DNA may play an important role in establishing chromosomalmosaicism in these organisms.

Several studies have shown that chronic NTHi infections displaysignificant genetic and phenotypic diversity (Loos, B. G., J. M.Bernstein, D. M. Dryja, T. F. Murphy, and D. P. Dickinson. 1989.Determination of the epidemiology and transmission of nontypeableHaemophilus influenzae in children with otitis media by comparison oftotal genomic DNA restriction fingerprints. Infect. Immun. 57:2751–2757;Porras, O., D. A. Caugant, B. Gray, T. Lagerigard, B. R. Levin, and C.Svanborg-Eden. 1986. Difference in structure between type; van Alphen,L., D. A. Caugant, B. Duim, M. O'Rourke, and L. D. Bowler. 1997.Differences in genetic diversity of nonencapsulated Haemophilusinfluenzae from various diseases. Microbiology 143:1423–1431, all ofwhich are incorporated by reference herein). van Alphen et al. usedmultilocus enzyme electrophoresis to examine the genetic diversity among80 isolates of NTHi recovered from both healthy patients and thosepresenting with different diseases. These researchers concluded thatchronic persistence in the host contributes to the genetic diversity ofNTHi.

To examine the degree of genomic plasticity among pathogenic strains ofH. influenzae, a highly redundant, pooled genomic library was generatedusing hydrodynamically sheared DNA from 10 clinical isolates culturedfrom pediatric patients undergoing treatment for chronic otitis mediawith effusion. This library is also being used as part of a largerinvestigation to study gene expression in order to delineate the geneticcontrol of biofilm formation in pathogenic strains of H. influenzae. Thepresent invention addresses the objective of evaluating the genomicplasticity between the nonpathogenic H. influenzae reference strain Rdand 10 clinical isolates, and DNA sequence evidence that reveals anextensive degree of plasticity among all strains.

The present invention involves isolated and cloned novel DNA fragmentsthat are present in pathogenic strains of Haemophilus influenzae(cultured from pediatric patients undergoing treatment for chronicotitis media with effusion) and are absent in strain Rd. Four of thesesequences are disclosed, whose corresponding amino acid sequencesdisplay varying degrees of homology to virulence-associated bacterialproteins.

SUMMARY OF THE INVENTION

The present invention pertains to a DNA sequence of Haemophilusinfluenzae clone 151_(—)04 shown in SEQ. ID. NO. 1.

The present invention pertains to a DNA sequence of Haemophilusinfluenzae clone 125_L2 shown in SEQ. ID. NO. 2.

The present invention pertains to a DNA sequence of Haemophilusinfluenzae clone 179_D 14 shown in SEQ. ID. NO. 3.

The present invention pertains to a DNA sequence of Haemophilusinfluenzae clone 167_A16 shown in SEQ. ID. NO. 4.

DETAILED DESCRIPTION

The present invention pertains to a DNA sequence of Haemophilusinfluenzae clone 151_(—)04 shown in SEQ. ID. NO. 1.

The present invention pertains to a DNA sequence of Haemophilusinfluenzae clone 125_L2 shown in SEQ. ID. NO. 2.

The present invention pertains to a DNA sequence of Haemophilusinfluenzae clone 179_D14 shown in SEQ. ID. NO. 3.

The present invention pertains to a DNA sequence of Haemophilusinfluenzae clone 167_A16 shown in SEQ. ID. NO. 4.

Referring more specifically to these DNA sequences:

(1) Clone 151_O4

Clone 151_O4 contains a novel DNA sequence found in every one of the 10pathogenic Haemophilus influenzae isolates. The most significant stretchof homology displayed by this novel nucleotide sequence is a stretch of110 bp with 76% homology to the related bacterium Pasteurella multocida(May et al., 2001). The full-length, putative protein encoded by thenovel 1337 bp sequence exhibits homology (47% identical, 68% similar) tothe YhbX/YhjW/YijP/YjdB family protein in the bacterial pathogenNeisseria meningitidis (Tettelin et al, 2000). This meningococcalprotein is named for its homology to a group of probablemembrane-associated proteins originally identified in E. coli. Mostinteresting is the E. coli YijP protein, which is involved in thepenetration of the blood-brain barrier and thereby contributes to thepathogenesis of E. coli meningitis (Wang et al., 1999). Also noteworthyis a tract of six copies of the tetranucleotide TITG that occurs withinthe first 150 nt of the putative open reading frame (ORF).Tetranucleotide repeats have been associated with virulence genes in H.influenzae (Hood et al., 1996, Ren et al., 1999).

Nucleotide sequence of H. influenzae clone 151_O4. The novel 1337 bpsequence that is being disclosed is underlined.

AGTAATATCACAATAGSGGATCCACGAGCTTCTATTAGGTATCGTATTGGCTGCAGAGGGATATCCAAAGGATTATCGCAAAGGCGATGAAATCAGCGGATTGCCTAAAAGTGCGGTCAAAAACGAGAAAGTTTTCTTAGCGGGTGTCGCAGAACAAGAAGGCAAGCTAGTCACAAACGGCGGTCGTGTACTTTGTGTGACTGCGTTAGGCGAAAGTGTATTTGAAGCACAACAAAAAGCGTTAAAATTGGCTGAGCAAATTCAATGGTCTGGGCGTTTTTATCGTCGAGACATTGGTTACAGGGCTGTGGAACGAGAACAAGCAAAATAGTTAGAAATCTTGTTTGAATTTAATTAGATAAAAAATATTGTACAGGGTAGAATTGTATTTTCCTAGGATTTAGGATTTTGTTAGGGCAACGTTTACGATTGCTCTGACAATAAATTAGAATTATTATTTTTGTTACTTTATGAGGTTATATCAACTTATGCGACAATTTGTCATCGTAGTATTATATTCTGCAATTCTTCTTTCATTAGAAGTTATTTATAGAAAATTATTTAATATTTCTAGTATTGAGAGATACACTGAAAGTTATTTGTCTGTTTGTTTGTTTGTTTGTTTGTTTGTTTTTTCAAAATATAGAATTACAAGAATATTAGTTGGCGCTTTATTTGCAATAAGTATTGTTGTTAATAATGTACATTATGCAGTATACCAATCTTGGATTGGACCTGTTAATTACTCACTTGCATTTAAAGAAATTAATGAGATAACAAATGCTGGCTTAACAATGATAGATAAATTCATATATCCATTGTTATTTGGTTTATTTGAAGTTGCTGTGTTTTTAAGTTTAAGTTTCATAAAAAGAAAAGTATATAAACTTTCTTGGATTTTTGACTTTATTTTTTATGCTGTGATGATGTATGTTTTTGTTCGAGCGTATACAACAAAATCCCATGAGCGTTTTATTTCACCTAACACTGTTTATTCTCGATTAAAATCCAATTATTTATCGTTGGGTTATTTTATAGGACGAATTGTTCCTTATGAGATATTTTCTTTATCTAATATTCCTCTTTATCATAAATCTAAGCCTATGAAATCGGGCTCTCCGVAAAATTMARGAATATAATTTTAATTAATGGGGGAAAGTGCGACCTCAAGTCATTTTAGTGCTTTTGGTTACGGGAGAAAAACATCTCCTTTTTTAGATAGCTTAAAATATAAATCAGGAGCTCTTGTTGGTAAAACTTATTCAGGAGGAAAGCTAACAGCAATTTCTTTACCAATGTTTTTTAATGCAATYCCTTAYCCAAATGGAATACAACAGATAGCTAAAGGAGATACGAATTTATTTAATTTAGCGAAAGAGCAAGGCTTTCAGACATATTTTTATTCAGCTCAAGCTAGGGATGATATGCATATGATCAATTTTTTAGGAGGAGCTTGGATTGATGATATTCGTTTTCCAGATAATGAAGGGTATTCTTTAAGAGATTCAATGCCTGATAATAAATTACTTCCTGCTTTTAAAAATATTAATTTAGATAATGGTTATCATTTTGTTGTTTTACATCATAGAGGGAGTCATATTCCCTATGGGGCATTATTAGAATGAAAAAGNAGNAAGGTGTTKGGAARAA ATAACG(2) Clone 125_L2

The 1,802-bp cloned H. influenzae fragment in 125_L2 is a novelpolynucleotide that is found in 9 of th 10 pathogenic Haemophilusinfluenzae isolates. Sequence similarity searches revealed only twoshort regions (approximately 75 bp each) that display nucleotidehomology (83%) to the Shigella resistance locus (SRL) pathogenicityisland (PAI) of S. flexneri serotype 2a. The 66,257-bp SRL PAI carriesgenes for antibiotic resistance, iron uptake, and at least 22prophage-related ORFs (Luck et al., 2001). The conceptual translation ofthe 125_L2 sequence and subsequent homology search revealed 3 openreading frames (one complete, two incomplete). All three inferredprotein products of this clone were homologous to the proteins encodedby ORFs 7, 8 and 9 of the SRL PAI (Luck et al., 2001). These Shigellahomologs included a probable LysR-like transcriptional regulator (ORF 7;64% identical, 73% similar), an unknown protein (ORF 8; 58%, 78%), and aputative anaerobic decarboxylate transporter (ORF 9; 54% identical, 64%similar).

Nucleotide sequence being disclosed for clone 125_L2.

CCTGCAACAGCTAGCTGAGATGATACAACCGATCCCGATAAAACTCTGGATGGTCTAATTCCAGATTCTTTTGCAACTTCAGCAATGACAGGTAATGTAGAAAAAACAATAAATCCAGTACCTGCAAAAATGGTCATTAACCAAGTAATCATTGGCGCAATAAAATTTATATGTTTGGGRTTTTTACGSATTAATCTTTCTGCATAATTAACTAAGRATGCCATTCCCCCTGTAGCTTGCAATGTTGCTGCTGRAAGRGRTACAGACATTATGRTCAAAATAACATCTATTGGRACAGRTCCTATTGGTAGTCTTAAACCCAACGTTAAGRTAGCAAGTCCAAGACCACCAAATAAACCAATGGCAATACCTCCAAATCTAAGGCCAAGTACAATAGAGGCTAGARCAACAAARATTTCAACCCMGACCATAATCATCTCCTTAATCAATGTTTGATACCAACGMATAGCAGCTCTAACTAATGCTGCTGTAGARTCCTCATAAAGTATAGGTTGCTCTCTCATTGCATTTTTTAGGATCARTGGGATTTCCGTACACCCTAGAATAATNACTTCTGAACCGTGACGAATAACTTCATCACGTTGTATTAACATTAATTCTTCAGCTTTTTGAATCTCTCCGCTCTTATATAAATAAATACTTTCCATGACCGATTTTTGATGTTCTTCATTGGGAAGAAGACAAATTAATTCCATATTTTTCTAATGTTTTCTGATATAGCTTTGTTGCTAAAGTAGCATCAGTAGCAAGAATACCAATCTTTGTTTTACCCATTTGTAGAACTTCATTAATTGTTGAATCAATAATATTTAACATATCAACATGACATTTCTCTTTTAGTTCATCATACCAATAATGTGCAGTATTACAGGCAATTAGGATACATTTAGCACCCGCATTTTCTAACCCATAAATGCGTTCCTCCATTGCAAGTAGTGGTGATTCTCCTCCATGCAAAATGGAAGTTGTACGATCGGGAATATCAGGAATAGACGAAATAACAAGAGGAATATGTTCTTGATCACAATGAGCTGGTGTAAATTGAATAAATTTCTGAAACATATCTGCCGTTGCTGCTGGTCCCATTCCACCTAAAATACCAATAATGTTCTTCATAAGAAAAATTCTCCTATTTATCTTTGGGTTATTTATTTTTAACAAAATCTAATGAAATAAGAGAAATGCAACAAATCGCACGCCAATGCAAATATTGCATAGCATAAATTGCGCACATTACAAATGTACAAAATAATGATTCAAATCAATATGATAAAAAACAAAAAGTGATAAGCTATTACATATTTAAGAATAAGGTATGCAAAATTAGCATAGAGAGAAATAATAAATGAAAAATATTGAAACAAAATGGTKAGAAGATBTTTTAATACTGGAAGATACACGCAATTTTTCACAGGCAGCAGAACATAGAAATTTATCGCAATCAGCTTTTAGCCGGAGAATAATTTCTCTTGAAGAATCTATTGGTGTAAAACYCTYCGATAGAYCCTCTGYCCCACTTCAACTTACAGAAGAAGGAAAATTATTTCATYCGCAAGHCTAGAAACCTTTTAAAACAGTTACAGTATAATCTTGATGAATTATTAGGGCAGAATACACAAAAAAACCGAATATAACTTTTGCAGCTGCACATTCCCTATCTTTATCTGTAATGCCAAAGTTAATTCATGATATTGGTCAATCACACCAGAACTTTATTTATTCCGTTGAAGCAATTGATGTTGATCAAACAGTAAAAACTTTGGTTGAAGGAAAAAGTGA(3) Clone 179_D14

This 950 bp polynucleotide is present in all 10 of the pathogenicHaemophilus influenzae isolates. It is highly homologous (88%) to thetrbB gene of the phytopathogen Ralstonia solanacearum, and its deducedamino acid sequence is 91% identical to the R. solanacearum probableconjugal transfer protein TrbB (Salanoubat et al., 2002). TrbB belongsto an extensive superfamily of proteins involved in the formation ofsurface-associated protein complexes that mediate a number of diverseprocesses such as pilus biosynthesis, DNA transport, and the secretionof virulence factors (Whitchurch et al, 1990; Hobbs and Mattick, 1993).This superfamily encompasses both archeal and bacterial proteins andincludes the subfamily of type IV NTPases to which TrbB proteins belong(Planet et al., 2001). Potentially noteworthy is the presence of apentanucleotide repeat (CCGGC) that repeats three times and is locatedwithin the putative open reading frame. The strong DNA homology of thisclone to R. solanacearum, and its extraordinarily high G+C (68%) contentcompared to that of Haemophilus DNA (38%), suggests that this DNA wasrecently acquired by H. influenzae.

Nucleotide sequence being disclosed for clone 179_D14.

TGGAAGTGATGCTCNACCCCGACCGGACATTGTGGGTGGATCGGCTGTCGTCTGGCCGTGCGCCGCTCGGCGTCGAACTGCCCGAAGCCGATGGCGAACGCATCATGGCGGTGGTCGCCGCCCATGTCGGTGCGGAGGTGCATCGCGGCCAACCGCTCTTGACCGCCGAACTGCCTGAAACCGGCGAACGCTTCGAGGGCATCCTGCCGCCCGCCGCACCCGGCCCGGCCCGGCCTTTGCGCTGCGCAAGCGTGCCGTGAGCATCATCGGTCTGGATCGCTATGTGGCTGATGGCATCCTGACCACTGGGCAGGCCGAGTTTCTGCGTCATGCCGTGCGCGAGCGGCACAACATCCTGATCGCCGGAGGCACCAGCACCGGCAAGACCACGCTGGCCAATGCCTTGCTGGCCGAGATCGCCGCCACCGGCGACCGCGTGCTGGTGCTCGAAGACACCATCGAACTGCAATGCGCGGCCCGCGACCATGTGCCGCTGCGCACCCGCGCCGGCGTCGTGTCCATGACCGAGCTGGTGCGGGCCACGATGCGCCTGCGGCCCGACCGCGTGATCGTCGGCGAAGTGCGCGGCGGCGAAGCGCTGGATCTGGTGAAGGTCTGGGGCACCGGCCACCCCGGCGGCATCGCCACCATTCATGCCGGCTCCGCGTTGGGCGCGCTGCTGCGCCTGGAGCAACTGATCCTCGAAGTGGCGGTGAATCCGCCCCGCGCCCTGATCGCCGAGGCGGTCAATGTCGTGATCCACATCGCAGGCCGCGGCCGCAAGCGCCACGTCGAAACCATTTCCCGCGTCGTCGGTTTCGACGGCGCGGGCTACCGCCTGGCGGATGCGCTGGAAGCGACGCTTCCCGAGCTGCCGCCGGTTCCTCTTACAGCCGCTGCCGCTACGCCTTCCTCGATCCCTGAACAACCTGGAGAACTGCCATGA CGCAClone 167_A16

The nucleotide sequence of clone 167_A16 is 2,597 nt in length and ispresent in 4 of our clinical H. influenzae strains. It displays twoshort regions of 55 and 60 nt with homology (96% and 90%, respectively)to Rd. The remaining sequence displays no significant homologies to anyknown DNA sequence. The proposed amino acid sequence suggests that thisclone encodes at least one protein with a tetratricopeptide repeat (TPR)motif. The tetratricopeptide repeat, which has been identified in a widevariety of proteins, is a structural motif that mediates protein-proteininteraction (Blatch and Lassle, 1999). The proteins with the highesthomologies (based on computer analysis) to the TPR protein encoded by167_A16 are almost exclusively from bacterial pathogens. Many of thesehomologs are, themselves, hypothetical proteins with unknown functions,including the Haemophilus somnus protein displaying the greatesthomology (42% identical; 60% similar). A few homologs, however, haveassigned functions that indicate the potential significance of thisprotein in H. influenzae. Included in these are the Legionellapneumophila enhanced entry protein (enhC gene product), which isinvolved in entry into host cells, and a Helicobacter pylori antigen(protein H) that granted significant protection against H. pylorichallenge in a mouse model system (Cirillo et al., 2000; Hocking et al,1999).

Downstream of the putative open reading frame for the TPR protein,another region was analyzed that appears to have a very weakly conservedTPR motif Its primary homolog is a conserved hypothetical H. pyloriprotein. Finally, the hypothetical translation of a third putativecoding region shows no evidence of a TPR motif and has its highesthomology to a Listeria monocytogenes protein that is similar to theputative integral membrane protein, ComEC, which is required for DNAuptake (Glaser, et al., 2001).

Nucleotide sequence being disclosed for clone 167_A16.

TTTGATTGAGCTCACGATATTTATCACACCCTTCTTGACTACGTAGATCGCAAGCCATGCCATAGTAAGATTTAGCTTTTTGCTCATCTTTATAAAGAAACGCGTTCCCTAATTCCACAAACACTGCAGGATCTTGGCTATTTTCCAATTCTAATTGCAATGTTTCAAATTTTGCTTTTACATCATCATTCGGCGCCTCATCTTTTAATTTTTTTAATTTTATTAACTTCACCTTTGTACCAATCACTGTCATTTCATCTTTAGTTTCTTTATTATGCTCTTGCAATAATTGCTCGGCTTTATTTTCATCTTTTACCGTGCCAATTCCCAAAATATAAAGAATAGCTAATTCACGATAAACGCTATTTGGACGAAATCGATTATTTTCTACTTGTCTAAACACCGCAGGTTGATGGTGTAATAGACTGCTTTTATAGGCTTTATCCAACCAATAAAAGCGGTTTTCCCAATCTGGTTTAATATTATCATTACCATCAAAATACCAACGCCCTAACTGTGCTTCCGCCATTGGATAACCATTATTTGCGGCTTGTTCCACCAACATATAACCTGTCAAAAAATCCTTATCCTTATTTACGGCATCTATAGMCAAGAATCATTTTGGCAAAATTATCGCCCGCATCCGCAGCCATTTTCATATAATGTTTTGACGATTCTTTATTTCCTTTGTCATTATAAATGGTTGCCAAACCACGATATGCCAACGGATAATTTTGATTGCTGGCTTTAAGAAACCACTCTGTCGCTAAATTTTTTTGACCTWTGATAAAATAATAACGCCCCAACTGATATTGCGTCACAGCATTGCCTTTTTCATGCCAACACTCGCAAACGTGCTGGAGAAAAATCTTCAAGTGCTTTNTCTAGCTTGCTGATCGCCATAATACTCCTGAGCACTAACTAATMVTTCTAGCTGTTTAATCTCACGATATTCTGGGTAATATTGGGTAAAATACACAGCTCCACCACCAATTACTGCCAATAATAAAACGGCTAAGGTTAATTTTTTCTTCATTATTTTGTTCCTTGATTTAATTGTTTATACATCTCACAACCTTTTTGCTCTTTATTATCACAAGCCTTGCCAAACCATTTTTTGGCAGTGGCAAAATTTTGTTTTACTCCTATTCCGCCCATATAAGCAAGACCAACTATTGCCTGCGCTCGAGAATTATTATTTTCTGCTGCTTTTTGATACCATTTTATGGCTTCAGTTTTATTTTCTTTTACTCCATCGCCATCATAATACATATCGCCCAATATCATTTGGGATTCAGTATCATTTTGATTTGCCGCTTTTTTCAACCATTTCACTGCTTCCGTATTATTCTGTTTTACGCCAACTCCATCTTTATACATCATTCCCACTTTAAATTGGGCATCAACATCATCTTGCTCCGCAGCTTCCTTCAACCATTTAAAGCCTTCTTGGTAATTTTGTTTTACGCCCAAGCCGTTAATATACATACCAGCTAAATCATATTGAGCGATACGTACACCTTGTTCAGCCGATTTTTTATACCATTTTATCGCTTCAAAATAATCTTGCTTTATGCCATCGCCATTTTTATATAACACCGCTAACATCCCCTGTGCAATCCCATCTCCCTGCTCTGCTAAAGGACGAATAATTGCTAATGCGGACTTAAAATCTTTCTGTTCAAATAAATGAACAATCTTATCAACTTGCTCCTCTTCCATTGCATAAACGGTTGATTGAAAAGAAAAGATAGAAGCACCGAAAAGTGCGGTGGTAAGAAGTGTTTTTGTTAGTTTCATTTTGTTTTCCTATTAAATTGAATGAATAAATAATCTTTTATTTTTATTCACTAAAATTGGCGTATAAGTAGAAAAATCTTTAAGTACTTCGCTATGTGGGTGCCCATTTCGTCGCTGGCTATCTGCTGAAAACACACTAAGACAAGGCGAAAATATGTTCGCTAATCCTTGCTGCCAATTATGCTTTGAACCGTGATGGGGAACTTGTAAGCMATMAATYSTSGCCATTCGTTCTACACCTAATGATTGCGTTAAATCGGTTAATAATGGCAAATCATTTAAAAACGCATCGCCTGTATATAAAATCGCATTTTTGTTTCTATCTTTTGGGAAAACAAGAATTTCATTTCCATCATTTTTAGGAATATCATAAATATAATTATTCCCTAGTCCCCAAACAGCTGTTGATGTTATATTTCTAATATATAAATATTGAGAAATGATATTTTTGTTTTTATTTCCATTTCCAAATGCGAGAGTATAGAGTGTTTTTAAGGCTGGCGTTGGATCTGTGGAATTAGACTGATGAGATTGAATAATTTGCTCAACCTGCTTTTGAAAAGCAGTCAAATTTGTTGGCACTTTTGCAAGCAAATGAAATGGCACGTTATATAAAACAAATTCAAACTGCTCTTCGCCTTTTCTAAATAAAAGGGCTTTATCAGGATTGAGCCAATGTACATTTTGTTTTAAGTTATCAAACTCATTTGATAATTTTTCAGTAGTTTTAAAAGAAAGTACATCATCAAAATTACTTGGTTCAAGAGTTATCAAAATTTCACTCTCATTTTCTTTTG

Since the genes that are responsible for the virulence of H. influenzaeare missing from strain Rd, the sequence of the Rd genome isinsufficient for developing tools to detect or prevent H. influenzaeinfections. The sequences identified herein were characterized based ontheir absence in strain Rd, their presence in pathogenic strains of H.influenzae, and their significant homologies (at the amino acid level)to proteins implicated in bacterial pathogenesis.

In the operation of the invention, ten strains of H. influenzae,cultured from pediatric middle-ear effusions at Children's Hospital ofPittsburgh, were obtained as pure first-plate isolates on chocolateagar. These strains, designated AA–JJ, were typed serologically usingslide agglutination at the Pittsburgh Public Health Laboratory. Allspecimens were tested initially with poly a–f antiserum (DifcoLaboratories, Detroit, Mich.). Specimens giving a positive reaction werethen tested with anti-a antiserum and anti-b antiserum. (Antiseraspecific for the less common serotypes c, d, e and f were not availableat this facility.) Strain AA was classified as an encapsulated strain ofserotype c, d, e, or f. The nine remaining clinical isolates (BB–JJ)were classified as nontypeable strains of H. influenzae.

Bacterial Growth Conditions. H. influenzae strains were grown in BrainHeart Infusion broth (Becton Dickinson, Sparks, Md.) supplemented withhemin (final concentration of 10 μg/ml; Fisher Scientific, Pittsburgh,Pa.), NAD (final concentration 2 μg/ml; Sigma, St. Louis, Mo.), andthiamine HCl (final concentration 20 μg/ml; Sigma) at 37° C. in a 5% CO₂atmosphere for one passage followed by storage in 22% glycerol at −80°C. E. coli TOP 10 cells was grown in Luria-Bertani broth or onLuria-Bertani agar (Becton Dickinson, Sparks, Md.) at 37° C. Kanamycin(Invitrogen, Carlsbad, Calif.) was added to a final concentration of 50μg/ml when necessary for selection.

Isolation of Bacterial Genomic DNA. Genomic DNA was extracted from eachclinical strain using a modification of the method described in Ausubelet al. (Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G.Seidman, J. A. Smith, and K. Struhl. 1990. Current protocols inmolecular biology. Greene Publishing Associates and Wiley-Interscience,New York, N.Y., incorporated by reference herein.) Cells were collectedby centrifugation from 100 ml overnight cultures and were resuspended inTE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0). The cells werelysed by the addition of sodium dodecyl sulfate (SDS; Invitrogen) to afinal concentration of 0.5% and incubated at 37° C. for one hour withRNAse A (final concentration 50 μg/ml; Gentra Systems, Inc.,Minneapolis, Minn.). Cellular proteins were removed with Proteinase K(final concentration 100 μg/ml; Invitrogen) at 37° C. for 1 h.Cetyltrimethylammonium bromide (CTAB; Sigma) was added to a finalconcentration of 1%, and samples were incubated at 65° C. for 20 min.Following a chloroform/isoamyl alcohol (24:1) extraction, the DNA wasprecipitated from the aqueous phase with 0.6 volumes of isopropanol. TheDNA was pelleted by centrifugation and washed with 70% ethanol. Afterair drying, the pellets were resuspended in TE buffer at 65° C. for 1 h.The samples were quantitated using UV spectrophotometry and theirquality analyzed by agarose gel electrophoresis.

Construction of pooled genomic library. A library of pooled genomic DNAwas constructed primarily to diminish the experimental bias that mighthave resulted from a single-strain library. The genomic DNA from eachclinical isolate was fragmented in the HydroShear™ (GeneMachines, SanCarlos, Calif.) by following the manufacturer's instructions forobtaining DNA fragments with an average length of 1.5 kb (range 1.0–2.5kb). Aliquots (10 μg each) of the sheared DNA preparations were pooled,end-repaired, ligated into the plasmid pCR®4Blunt-TOPO and transformedinto E. coli TOP10 all according to the manufacturer's protocol(Invitrogen, Corp., Carlsbad, Calif.). A detailed description of thelibrary construction is presented elsewhere (Erdos, G. S., S. Sayeed, P.Antalis, F. Z. Hu, J. Hayes, J. Goodwin, R. Dopico, J. C. Post, and G.D. Ehrlich. 2003. Development and characterization of a pooledHaemophilus influenzae genomic library for the evaluation of geneexpression changes associated with mucosal biofilm formation in otitsmedia. Int. J. Pediatr. Otorhinolaryngol., 67:749–755, incorporated byreference herein). Briefly, the Q-bot 3-Axis XYZ Multi-Tasking Robot(Genetix Limited, UK) was used to array 76,800 transformants toconstruct the pooled H. influenzae library, which was stored in 10%glycerol at −80° C. Clones in the library were chosen randomly forfurther analysis.

DNA sequencing. Plasmid DNA templates were prepared for sequencing usingthe QIAprep Miniprep Kit (Qiagen, Inc., Valencia, Calif.). Prior tosequencing, plasmid preparations were digested with EcoRI (Invitrogen)and analyzed on ethidium bromide-stained 1% agarose gels in TAE buffer.Only those constructs containing insertions of H. influenzae DNA thatappeared to be larger than 0.5 kb were used as sequencing templates. TheLiCor IR² Gene ReadIR™ and the Beckman Coulter CEQ 2000 XL automatedfluorescence sequencing systems were used to sequence the clones.

Sequencing reactions for the LiCor IR² Gene ReadIR™ DNA Analysis Systemwere prepared according to the Excel II Simultaneous Bi-DirectionalCycle Sequencing protocol provided by Li-Cor, Inc. (Lincoln, Nebr.).SeqiTherm Excel II DNA Sequencing Kits were purchased from EpicentreTechnologies (Madison, Wis.). Fluorescent dye-labeled M13 and T7 primers(Table 1) were synthesized by L1-Cor, Inc. Sequencing reactions werecarried out in Perkin Elmer 9600 thermal cyclers and included an initial2-minute denaturation step at 94° C., followed by 40 cycles with a 30-sdenaturation step at 92° C., a 15-s primer annealing step at 55° C., andan extension step of 15 s at 70° C. The final cycle was followed bystorage at 4° C. Sequencing gels were prepared using 6% Long Ranger gelsolution (BioWhittaker Molecular Applications, Rockland, Me.), 7M urea(Invitrogen), and 1.2×TBE (Invitrogen). Sequences were culled with BaseImagIR™ V.4.0 computer software (Li-Cor).

Sequence reactions for the Beckman Coulter CEQ 2000 XL DNA AnalysisSystem were prepared using the CEQ 2000 Dye Terminator Cycle Sequencingwith Quick Start Kit (Beckman Coulter, Inc., Fullerton, Calif.). Bothunlabeled M13 and T7 primers, and specific primers for unique internalsequences of the H. influenzae cloned fragments were synthesized(Invitrogen) (Table 1). Cycling conditions were as follows: 30 cycleswith a 20-s denaturation step at 96° C.; a 20-s annealing step at 50°C.; an extension step of 2 min at 60° C.; and a hold at 4° C. Ethanolprecipitation was performed in an Allegra-25R centrifuge.

DNA sequence analysis. Sequences were analyzed and contig sequences wereformed using Sequencher version 4.0.5 software package (Gene CodesCorporation, Ann Arbor, Mich.). DNA sequence similarity searches usingthe basic BLASTn and BLASTx algorithms (Altschul, S. F., W. Gish, W.Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignmentsearch tool. J. Mol. Biol. 215:403–410, incorporated by referenceherein) were performed at the National Center for BiotechnologyInformation website (http://www.ncbi.nlm.nih.gov/).

Codon usage analysis of nonRd sequences. Published codon usage tableswere obtained from the Kazusa DNA Research Institute website(http://www.kazusa.or.jp/codon/) (Nakamura, Y., T. Gojobori, and T.Ikemura. 2000. Codon usage tabulated from the international DNA sequencedatabases: status for the year 2000. Nucl. Acids Res. 28:292,incorporated by reference herein). The least squares optimization method(Cox, S. R., and D. E. Williams. 1981. Representation of the molecularelectrostatic potential by a net atomic charge model. J. Comput. Chem.2:304–323, incorporated by reference herein) was applied to thesequences of a set of genes from the Haemophilus Rd genome to confirmthat it could distinguish Haemophilus-like codon usage from that ofother organisms (J. Gladitz et al., manuscript in preparation). Thismethod was then used to fit the codon usage of putative reading framesto the reported codon usage of the 71 organisms listed in Table 2. Theseparticular organisms, which include prokaryotes, eukaryotes, phage andviruses, were selected on the basis that their genomes provided acontinuous spectrum of G+C contents ranging from 24.62% to 67.67% (Table2). In addition, many of these organisms encode proteins that displayhomology to the conceptual translations of our nonRd sequences.

The above process involved optimizing a scaling parameter for each aminoacid in order to ‘best fit’ the codon usage of our reading frames to thereported codon usage of the corresponding amino acid in the 71representative organisms. The minimized sum of the squared differences(equation 1) was used as a measure of the similarity of codon usage forall tested amino acids:

$\begin{matrix}{ɛ_{A} = {\sum\limits_{i = 1}^{n}\;\left( {f_{i,A} - {C_{A}*g_{i,A}}} \right)^{2}}} & (1)\end{matrix}$in which f_(i,A) represents the percent usage of the i^(th) codon ofamino acid A in the reading frame being tested, g_(i,A) is the percentusage of the i^(th) codon of amino acid A in the organism being testedagainst, C_(A) is the optimization parameter used as a fitting factorfor amino acid A, and n is the number of codons existing for amino acidA. These individual amino acid measures (∈) were summed (equation 2) toproduce an overall measure of fit (F) for the analyzed reading frame:

$\begin{matrix}{F = {\sum\limits_{1}^{m}\; ɛ_{A}}} & (2)\end{matrix}$in which m is the number of different codons used for amino acid A.

The use of a single optimization parameter (C_(A)) per amino acidpreserved the pairwise codon ratios existing within each amino acid ofthe organism being tested while simultaneously adjusting for anyfrequency of use differences that an amino acid might have in ourreading frame versus its average usage in a given, much larger genome.The amino acids methionine and tryptophan, which use only one codoneach, always generate a zero value for ∈ and thus do not have an effectin this analysis.

Availability of nucleotide sequences. The novel (nonrd) nucleotidesequences identified herein will be made available on a website.

PCR-based Gene Distribution Studies. Primer pairs designed to obtaininternal sequence data for each H. influenzae clone were also used forPCR-based distribution studies in which genomic DNA isolated from eachclinical strain and from the laboratory reference strain Rd were used astemplates to determine the number of clinical strains that possessedeach nonRd sequence. A positive control reaction for each genomictemplate DNA was also performed using primers specific for the H.influenzae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (Table1). PCR was performed using the Eppendorf MasterTaq Kit (BrinlananInstruments, Inc., Westbury, N.Y.) in a 25 μl reaction mixture. Standardreactions included 0.6 units of Taq DNA polymerase, 50 ng of templateDNA, 20 pmol of each primer, 1.5 mM MgCl₂, and 0.2 mM dNTPs. Reactionswere carried out in Perkin Elmer 9600 thermal cyclers and included aninitial 10-minute denaturation step at 95° C. The amplification cycleconsisted of 30 s at 94° C., 1 min at 55° C., and 1 min at 72° C. for 35cycles. This was followed by a final extension step of 7 min at 72° C.and then a 4° C. hold. Reactions were analyzed on 1.7% agarose gels thatwere stained with ethidium bromide.

Redundancy of the pooled genomic library. The library of pooled genomicDNA from the ten clinical H. influenzae strains was comprised of 76,800clones. Accounting for the approximately 25% of clones havingoligonucleotide inserts or low viability (data not shown), thefunctional library contained approximately 57,000 clones. The averageinsert size of the library clones was 1.5 kb, resulting in a librarythat consisted of approximately 8.55×10⁷ bp of DNA from the 10 clinicalstrains. Since the genome size of H. influenzae is approximately 1.83 Mb(Fleischmann, R. D., M. D. Adams, O. White, R. A. Clayton, E. F.Kirkness, A. R. Kerlavage, C. J. Bult, J.-F. Tomb, B. A. Dougherty, J.M. Merrick, K. McKenney, G. Sutton, W. FitzHugh, C. Fields, J. D.Gocayne, J. Scott, R. Shirley, L.-I. Liu, A. Glodek, J. M. Kelley, J. F.Weidman, C. A. Phillips, T. Spriggs, E. Hedblom, M. D. Cotton, T. R.Utterback, M. C. Hanna, D. T. Nguyen, D. M. Saudek, R. C. Brandon, L. D.Fine, J. L. Fritchman, J. L. Fuhrmann, N. S. M. Geoghagen, C. L. Gnehm,L. A. McDonald, K. V. Small, C. M. Fraser, H. O. Smith, and J. C.Venter. 1995. Whole genome random sequencing and assembly of Haemophilusinfluenzae Rd. Science 269:496–512, incorporated by reference herein),the redundant library provided a 4.6×coverage of each of the 10 genomes.

Comparison of DNA sequences to the H. influenzae Rd genome. Thenucleotide sequences of 771 randomly chosen clones from the pooled H.influenzae library were analyzed. This set of sequenced clonesrepresented 1.35% of the functional library. The clones were sequencedwith both forward and reverse primers, with an average read length of650 bases obtained at each end for each clone. This provided us withapproximately 87% of the complete sequence for the average-sized (1.5kb) clone. The sequences obtained were compared to that of the publishedsequence of the H. influenzae Rd reference genome to identify thosenovel sequences present in the set of clinical isolates but absent inthis less virulent laboratory reference strain.

Clones displaying at least 350 bp of contiguous homology to Rd at eachend were classified as Rd-like. To ensure that there were no largedeletions or insertions in these sequences, the size of each clone(determined by restriction endonuclease analysis) was compared to thesize of the corresponding region on the Rd genome. Using this method,699 (90.7%) of the clones as Rd-like sequences were classified.

However, it is possible that this value is deceptively high, since theunsequenced regions of many of the clones could contain subtle,unidentified deviations from the respective Rd sequences that were notdetected by electrophoretic sizing of the insert. In fact, possiblesmall insertions and deletions in several of the Rd-like clones werealready detected, and the detailed analysis and verification of thesechanges are in progress and part of future studies.

Identification of novel (non rd) DNA sequences. BLASTn analysis of theinitial sequences obtained for 72 clones (9.3% of the total number ofclones analyzed) indicated that these clones either varied significantlyor were absent entirely from the Rd genome. Complete double-strandedsequence was then obtained, and a consensus sequence was assembled, foreach of these clones. The consensus sequences of these 72 clones werethen compared to each other to determine if any sequence occurred inmore than one clone. Each set of overlapping consensus sequences werecompiled into a contig sequence, resulting in the formation of ninecontigs using the sequences of 21 clones. These nine contig sequences,in addition to the consensus sequences of the 51 remaining clones, wereused to perform additional nucleotide (BLASTn) and amino acid (BLASTx)homology searches. Of these 60 sequences, 40 displayed nonucleotide-level homology to the Rd genome. The remaining 20 sequencescontained varying lengths of homologous regions to Rd in addition tosignificant (≧200 bp) regions not present in Rd.

Distribution of cloned Rd-like sequences on the Rd genome. FIG. 1 showsthe regions of the Rd genome represented in the cloned sequences.Sequences from the entire Rd chromosome were distributed evenly in theset of clones, suggesting that the clones did not overrepresent a singlepathogenicity island per se. The equal distribution of randomly selectedclones along the H. influenzae Rd chromosome indicated that theredundant pooled genomic library was not degraded and was unbiased inits coverage of the ten clinical isolates. Additionally, the 20sequences that exhibited varying degrees of Rd homology were alsodistributed evenly throughout the Rd chromosome.

Distribution of novel sequences among the ten OM isolates. Primer pairs(Table 1) flanking nonRd sequences were used in PCR-based studies todetect the presence or absence of the novel nucleotide sequences in thegenomes of the 10 H. influenzae clinical strains and also in Rd. Eachprimer pair was tested initially using plasmid DNA from the respectiveclone as the template. The primer pairs that supported amplificationusing the corresponding plasmid clones were further used in the genomicdistribution study. The presence of a PCR product at the predictedmolecular weight following agarose gel electrophoresis was taken asevidence that the novel sequence was present in the genome of theclinical isolate.

Data for these genomic distribution studies were summarized in Table 3.If the distribution patterns were the same for clones that formed acontig sequence, distribution pattern was reported only once. In a fewcases, the individual distribution patterns for overlapping clones werereported because each provided unique information. The mean distributionof the unique sequences was 8.48±2.55 strains. The mode and the medianwere both equal to 10. None of the ten clinical strains harbored thesame set of unique sequences. Interestingly, the distribution of thenonRd sequences in the encapsulated strain AA did not appear to besubstantially different from those of the nine NTHi isolates.

Occurrence of tandem oligonucleotide repeat sequences. The novelsequences were searched for tandem repeats of oligonucleotides rangingin length from two to 17 bases. Variable-number-of-tandem-repeat regions(VNTRs) in H. influenzae and other pathogenic bacteria are associatedwith phenotypic switching and virulence (Hood, D. W., M. E. Deadman, M.P. Jennings, M. Bisercic, R. D. Fleischmann, J. C. Venter, and E. R.Moxon. 1996. DNA repeats identify novel virulence genes in Haemophilusinfluenzae. Proc. Natl. Acad. Sci USA. 93:11121–11125; van Belkum, A.,S. Scherer, W. van Leeuwen, D. Willemse, L. van Alphen, and H. Verbrugh.1997. Variable number of tandem repeats in clinical strains ofHaemophilus influenzae. Infect. Immun. 65:5017–5027, both of which areincorporated by reference herein). Five VNTRs were identified, each in adifferent clone: a mononucleotide repeat (121_L20); two tetranucleotiderepeats (151_O4 and Hb_contig); a pentanucleotide repeat (179_D14); andan 11-mer repeat (162_D23). The mononucleotide repeat in 121_L20, whichhad a G+C content of only 32%, consisted of 12 G-residues. The remainingVNTR sequences, and their potential functions, are presented below inthe context of the respective clones.

Occurrence of USSs in unique clones. Each of the nonRd sequences werealso searched for the presence of the Haemophilus and Neisseria USSs.Bacteria belonging to both of these genera are naturally competent andpreferentially take up DNA containing their respective USS. TheHaemophilus uptake sequence (hUSS) consists of a conserved 9-bp coresequence contained within a 29-bp sequence5′-aAAGTGCGGTnRWWWWWnnnnnnRWWWWW-3′ (Danner, D. B., R. A. Deich, K. L.Sisco, H. O. Smith. 1980. An eleven-base-pair sequence determines thespecificity of DNA uptake in Haemophilus transformation. Gene11:311–318; Goodgal, S. H., and M. A. Mitchell. 1990. Sequence anduptake specificity of cloned sonicated fragments of Haemophilusinfluenzae DNA. J. Bacteriol. 172:5924–5928; Smith, H. O., J.-F. Tomb,B. A. Dougherty, R. D. Fleischmann, and J. C. Venter. 1995. Frequencyand distribution of DNA uptake signal sequences in the Haemophilusinfluenze Rd genome. Science 269:538–540, all of which are incorporatedby reference herein). The neisserial sequence (nUSS) is the 10-bpsequence 5′-GCCGTCTGAA-3′ (Elkins, C., C. E. Thomas, H. S. Seifert, andP. F. Sparling. 1991. Species-specific uptake of DNA by gonococci ismediated by a 10-base-pair sequence. J. Bacteriol. 173:3911–3913;Goodman, S. D., and J. J. Scocca. 1988. Identification and arrangementof the DNA sequence recognized in specific transformation of Neisseriagonorrhoeae. Proc. Natl. Acad. Sci. U.S.A. 85:6982–6986, both of whichare incorporated by reference herein). 32 hUSSs and 2 nUSSs distributedamong 28 genomic clones (Table 4) were identified. No cloned fragmentcontained both an hUSS and an nUSS, although several contained more thanone hUSS.

Nucleotide homologies displayed by nonRd sequences. BLASTn analysis ofthe 60 nonRd sequences listed in Table 4 revealed that only 18 hadstrong homology to a known nucleotide sequence. 14 of these 18 werehomologous to DNA from pathogenic strains of H. influenzae. TheFPG_contig and 97_H3 showed strong nucleotide homology (97% and 81%,respectively) to DNA from the closely related pathogenic bacteriumPasteurella multocida. Clone 100_E23 was 99% homologous to H. influenzaephage HP2, and 179_D14 was 88% homologous to DNA from the plant pathogenRalstonia solanacearum.

Three-frame, forward and reverse, BLASTx analysis were then performed todetermine if the conceptual protein translations of the nonRd sequencesdemonstrated homology to any known proteins. Given that random clonesordinarily code for only partial open reading frames (ORFs), the BLASTxanalysis frequently involved the comparison of partial protein sequencesto the full-length sequences in the protein databases. To aid in theinterpretation of the reported homologies, and to avoid themisconception that a full-length ORF was always analyzed, eachtranslated sequence as a region of homology (ROH) was defined. An ROHmay have contained stop codons or frameshifts that were trangressed infavor of continuing a strong homology. Multiple ROHs for the majority ofthe clones were analyzed. Table 4 summarizes the data obtained from theextensive sequence analysis and homology searches using the nonRdsequences. For each ROH, its length (in amino acids) along with thelength of its primary protein homolog was reported. Homologies aredescribed in more detail below.

Homologies to H. influenzae virulence factors. 20% of the nonRd clonesthat were analyzed exhibited homology (both nucleotide and amino acid)to H. influenzae virulence factors. One fourth of this subset washomologous to the H. influenzae fimbrial gene cluster (hij), which ismissing in Rd (van Ham, S. M., L. van Alphen, F. R. Mooi, and J. P. M.van Putten. 1994. The fimbrial gene cluster of Haemophilus influenzaetype b. Mol. Microbiol. 6:277–282, incorporated by reference herein).Three clones were homologous to the two major adherence proteins ofnontypeable H. influenzae isolates, the high-molecular-weight (HMW)surface-exposed proteins HMW1 and HMW2 (Barenkamp, S. J., and E.Leininger. 1992. Cloning, expression, and DNA sequence analysis of genesencoding nontypeable Haemophilus influenzae high-molecular-weightsurface-exposed proteins related to filamentous hemagglutinin ofBordetella pertussis. Infect. Immun. 60:1302–1313, incorporated byreference herein).

Clone 135_I10 was homologous to the putative virulence-associatedautotransporter protein, Las, from H. influenzae biogroup aegyptius.Autotransporters are outer membrane proteins (OMPs) involved in theestablishment and dissemination of infection and include H. influenzaeLav, N. meningitidis VapA and Bordetella pertussis PerT (Davis, J., A.L. Smith, W. R. Hughes, and M. Golomb. 2001. Evolution of anautotransporter: domain shuffling and lateral transfer from pathogenicHaemophilus to Neisseria. J. Bacteriol. 183:4686–4635, incorporated byreference herein). lav is a mobile contingency gene located within apair of hUSSs (J. Davis, et al., 2001), and 135_I10 did contain an hUSS,but it occurred within the putative autotransporter ORF.

Two clones (9_E14 and 13_D9) that were homologous to the tryptophanasegenes tnaA and tnaB from Hib strain Eagan (Martin, K., G. Morlin, A.Smith, A. Nordyke, A. Eisenstark, and M. Golomb. 1998. The tryptophanasegene cluster of Haemophilus influenzae type b: evidence for horizontalgene transfer. J. Bacteriol. 180:107–118, incorporated by referenceherein) were compiled to form the Tna_contig sequence. The ability tocatabolize tryptophan is strongly associated with virulence, and the tnacluster appears to act as a pathogenicity island (K. Martin et al.,1998). Genomic DNA from all 10 of the OM isolates supported PCR with atnaB-specific primer set (see 9_E14 in Table 3). However, resultsgenerated with a primer pair including a tnaA-specific primer impliedthat only seven of the clinical strains had the tryptophanase structuralgene tnaA (see 13_D9 in Table 3).

Clone 83_M12 contained the entire lex2A ORF and the 5′ region of lex2Bfrom Hib strain DL42. The Lex2B_contig (formed from clones 47_C18 and93_M17) contained the 3′ end of lex2B and did not overlap with 83_M12.The lex2AB genes are involved in the variable expression oflipooligosaccharide (LOS) epitopes, which results in alterations invirulence (Cope, L. D., R. Yogev, J. Mertsola, J. L. Latimer, M. S.Hanson, G. H. McCracken, Jr. and E. J. Hansen. 1991. Molecular cloningof a gene involved in lipooligosaccharide biosynthesis and virulenceexpression by Haemophilus influenzae type B. Mol. Microbiol.5:1113–1124; Foxwell, A. R., J. M. Kyd, and A. W. Cripps. 1998.Nontypeable Haemophilus influenzae: pathogenesis and prevention.Microbiol. Mol. Biol. Rev. 62:294–308; Jarosik, G. P., and E. J. Hansen.1994. Identification of a new locus involved in expression ofHaemophilus influenzae type b lipooligosaccharide. Infect. Immun.62:4861–4867, all of which are incorporated by reference herein). Thelex2A allele contained only three repeating units of the tetranucleotideGCAA, which occurs 18 times in the 5′ region of lex2A in strain DL42(Jarosik et al., 1994). The sequence 3upstream of lex2A in 83_M12 washomologous to H. influenzae Rd purL. This nucleotide homology endedabruptly where the homology to the lex2AB locus began. A 15-bp sequence(5′-CAGATTTTCACTGTG-3′) was identified that is present both downstreamof the Rd purL and upstream of the lex2A in strain DL42 and whichprobably facilitated the insertion of the lex2 genes. A hUSS 10 bpupstream of this 15-bp sequence was also identified, indicating thatthis region of DNA would be preferentially taken up via transformationinto H. influenzae.

Homologies to H. influenzae hemoglobin-binding proteins. The sequencesof three overlapping clones (101_K4, 131_L20, and 153_C10) to form oneHb_contig sequence of 4,518 bp were complied, which provided a morecomprehensive view of this locus. Over half (>2,350 bp) of this contigsequence was novel with respect to known sequences in the publicdatabases, although each end was highly homologous (>90%) to hgpA fromHib strain HI689 (Jin, H., Z. Ren, J. M. Pozsgay, C. Elkins, P. W.Whitby, D. J. Morton, and T. L. Stull. 1996. Cloning of a DNA fragmentencoding a heme-repressible hemoglobin-binding outer membrane proteinfrom Haemophilus influenzae. Infect. Immun. 64:3134–3141; Jin, H., Z.Ren, P. W. Whitby, D. J. Morton, and T. L. Stull. 1999. Characterizationof hgpA, a gene encoding a hemoglobin/hemoglobin-haptoglobin-bindingprotein of Haemophilus influenzae. Microbiology 145:905–914, both ofwhich are incorporated by reference herein). hgpA encodes a protein thatbinds both hemoglobin and the hemoglobin-haptoglobin complex (Jin etal., 1999). BLASTx analysis of our contig revealed a complete ORF withcomparable homologies to a number of H. influenzae hemoglobin-bindingproteins, including the gene products of hgpA, hgpB, hgpC, hhuA.

Like many of these genes that encode OMPs involved in hemoglobinbinding, the putative ORF contained a series of CCAA nucleotide repeatsnear the N-terminus (Fleischmann, et al., 1995; Hood et al., 1996; Jinet al., 1999; Maciver, I., J. L. Latimer, H. H. Liem, U.Muller-Eberhard, Z. Hrkal, and E. J. Hansen. 1996. Identification of anouter membrane protein involved in utilization of hemoglobin-haptoglobincomplexes by nontypeable Haemophilus influenzae. Infect. Immun.64:3703–3712, all of which are incorporated by reference herein). Ren etal. (Ren et al., 1999) have shown that changes in the number of CCAArepeats in hgpA mediate phase variable expression. Anothercharacteristic that ORF shares with hgpA is the presence of a directrepeat, with unknown function, upstream of the putative start codon (Jinet al., 1996).

The sequence downstream of the cloned hgpA-like ORF suggested that thislocus is located in the same region of its respective genome as hgpA instrain HI689. The HI689 hgpA is located upstream of pepE, in place ofORFs HI0588, HI0589, HI0590, HI0591 and HI0592, which are upstream ofpepE on the Rd genome (Jin et al., 1999). Approximately 500 bpdownstream of our putative hemoglobin-binding ORF was the start of asecond, incomplete ORF that displayed 98% identity to the correspondingregion of the Rd pepE.

Clone 32_B2 also displayed some homology to H. influenzaehemoglobin-binding proteins. Of the 1,199 bp in this clone, only aninternal region of 332 bp displayed significant nucleotide-levelhomology (91%) to any sequences in the public databases. This homologywas to the 3′ end of hhuA from the nontypeable H. influenzae strainTN106 (Maciver et al., 1996). In addition, the theoretical translationof 32_B2 was 57% identical to the final 191 amino acids of thehemoglobin-haptoglobin binding protein HhuA (Table 4). These resultssuggested that 32_B2 contained the 3′ end of an incomplete ORF, which iseither a newly identified allele of hhuA or which encodes anotherhomolog to the group of H. influenzae proteins involved in the uptake ofheme.

Homology to Pasteurella multocida formamidopyrimidine DNA glycosylase(FPG). The entire 1.66 kb Fpg_contig sequence (compiled from clones112_A12 and 134_O6) was 97% homologous to the P. multocida fpg, whichencodes formamidopyrimidine-DNA glycosylase. Only three dispersedregions (≦70 nt each) were homologous to the Rd genome, despite the factthat Rd has an fpg gene. The presence of two hUSSs, one within the P.multocida fpg and the other downstream, indicated that this PasteurellaDNA had probably been taken up and incorporated into H. influenzae byway of transformation. Eight of our isolates were shown to harbor thisP. multocida fpg locus (Table 3).

Homology to the N. meningitidis YhbX/YhjW/YijP/YjdB family protein. Thepredicted amino acid sequence for the novel sequence in clone 151_O4exhibited greatest homology (47% identical, 68% similar; Table 4) to thehypothetical YhbX/YhjW/YijP/YjdB family protein in Neisseriameningitidis (Parkhill J., M. Achtman M, K. D. James, S. D. Bentley, C.Churcher, S. R. Klee, G. Morelli, D. Basham, D. Brown, T. Chillingworth,R. M. Davies, P. Davis, K. Devlin, T. Feltwell, N. Hamlin, S. Holroyd,K. Jagels, S. Leather, S. Moule, K. Mungall, M. A. Quail, M. A.Rajandream, K. M. Rutherford, M. Simmonds, J. Skelton, S. Whitehead, B.G. Spratt, B. G. Barrell. 2000. Complete DNA sequence of a serogroup Astrain of Neisseria meningitidis Z2491. Nature 404:502–506; 60,incorporated by reference herein). This meningococcal protein is namedfor its homology to a group of probable membrane-associated proteinsoriginally identified in E. coli. Most interesting is the E. coli YijP,which is involved in the penetration of the blood-brain barrier and hasbeen demonstrated to contribute to the pathogenesis of E. colimeningitis (Wang, Y., S.-H. Huang, C. A. Wass, M. F. Stins, and K. S.Kim. 1999. The gene locus yijP contributes to Escherichia coli K1invasion of brain microvascular endothelial cells. Infect. Immnun.67:4751–4756, incorporated by reference herein).

Genomic PCR distribution studies revealed that all ten otitis mediaisolates contain the novel sequence cloned in 151_O4 (Table 3). Thiswidespread distribution may be due to the presence of an hUSS 375 bpupstream of the predicted ORF. Also noteworthy is a tract of six copiesof the tetranucleotide TTTG within the first 150 nt of the putative ORF.Tetranucleotide repeats in the 5′ ends of H. influenzae reading frameshave been associated with slipped-strand mispairing, resulting in frameshifts that lead to phase-variable expression of a number of surfaceprotein genes (Davis, J., A. L. Smith, W. R. Hughes, and M. Golomb.2001. Evolution of an autotransporter: domain shuffling and lateraltransfer from pathogenic Haemophilus to Neisseria. J. Bacteriol.183:4686–4635; Hood, D. W., M. E. Deadman, M. P. Jennings, M. Bisercic,R. D. Fleischmann, J. C. Venter, and E. R. Moxon. 1996. DNA repeatsidentify novel virulence genes in Haemophilus influenzae. Proc. Natl.Acad. Sci USA. 93:11121–11125; Ren, Z., H. Jin, P. W. Whitby, D. J.Morton, and T. L. Stull. 1999. Role of CCAA nucleotide repeats inregulation of hemoglobin and hemoglobin-haptoglobin binding proteingenes of Haemophilus influenzae. J. Bacteriol. 181:5865–5870, all ofwhich are incorporated by reference herein).

Clonal similarity to a Shigella flexneri pathogenicity island. The1,802-bp H. influenzae fragment cloned in 125_L2 is a novelpolynucleotide that was detected in 9 of the 10 pathogenic Haemophilusinfluenzae isolates (Table 3). Sequence similarity searches revealed twoshort regions (approximately 75 bp each) that displayed nucleotidehomology (83%) to the Shigella resistance locus (SRL) pathogenicityisland (PAI) of S. flexneri type 2a. The 66,257-bp SRL PAI carries genesfor antibiotic resistance, iron uptake, and at least 22 prophage-relatedORFs (Luck, S. N., S. A. Turner, K. Rajakumar, H. Sakellaris, and B.Adler. 2001. Ferric dicitrate transport system (Fec) of Shigellaflexneri 2a YSH6000 is encoded on a novel pathogenicity island carryingmultiple antibiotic resistance genes. Infect. Immun. 69:6012–6021,incorporated by reference herein). The conceptual translation of the125_L2 sequence and subsequent homology search revealed 3 open readingframes (one complete, two incomplete). All three inferred proteinproducts of this clone were homologous to the proteins encoded by ORFs7, 8 and 9 of the SRL PAI (Luck, S. N., S. A. Turner, K. Rajakumar, H.Sakellaris, and B. Adler. 2001. Ferric dicitrate transport system (Fec)of Shigella flexneri 2a YSH6000 is encoded on a novel pathogenicityisland carrying multiple antibiotic resistance genes. Infect. Immun.69:6012–6021, incorporated by reference herein). These Shigella homologsincluded a probable LysR-like transcriptional regulator (ORF 7; 64%identical, 73% similar), an unknown protein (ORF 8; 58%, 78%), and aputative anaerobic decarboxylate transporter (ORF 9; 54% identical, 64%similar).

Presence of a tetratricopeptide repeat (TPR) protein. The nucleotidesequence of clone 167_A16, which was 2,597 nt in length, displayed onlytwo, short regions of 55 and 60 nt with homology (96% and 90%,respectively) to Rd. The remaining sequence displayed no significanthomologies to any known DNA sequence. The proposed amino acid sequencesuggested that this clone encodes at least one protein with atetratricopeptide repeat (TPR) motif The tetratricopeptide repeat, whichhas been identified in a wide variety of proteins, is a structural motifthat mediates protein-protein interactions (Blatch, G. L., and M.Lassle. 1999. The tetratricopeptide repeat: a structural motif mediatingprotein-protein interactions. Bioessays 21:932–939, incorporated byreference herein). The proteins with the highest homologies to ourhypothetical TPR protein (167_A16 ROH2, Table 4) are almost exclusivelyfrom bacterial pathogens. Many of these homologs are, themselves,hypothetical proteins with unknown functions, including the Haemophilussomnus protein displaying the greatest homology (42% identical; 60%similar). A few homologs, however, have assigned functions that lead usto speculate on the potential significance of this protein in H.influenzae. Included in these are the Legionella pneumophila enhancedentry protein (enhC gene product), which is involved in entry into hostcells, and a Helicobacter pylori antigen (protein H) that grantedsignificant protection against H. pylori challenge in a mouse modelsystem (Cirillo, S. L., G., J. Lum, and J. D. Cirillo. 2000.Identification of novel loci involved in entry by Legionellapneumophila. Microbiology 146:1345–1359; Hocking, D., E. Webb, F.Radcliff, L. Rothel, S. Taylor, G. Pinczower, C. Kapouleas, H. Braley,A. Lee, and C. Doidge. 1999. Isolation of recombinant protectiveHelicobacter pylori antigens. Infect. Immun. 67:4713–4719, both of whichare incorporated by reference herein).

Another region (ROH1) was analyzed, downstream of the putative ORF forthe TPR protein, that appeared to have a very weakly conserved TPRmotif. Its primary homolog was a conserved hypothetical H. pyloriprotein. Finally, the conceptual translation of a third putative codingregion (ROH3) showed no evidence of a TPR motif and had greatesthomology to a Listeria monocytogenes protein that is similar to theputative integral membrane protein, ComEC, which is required for DNAuptake in bacteria (Glaser, P., L. Frangeul, C. Buchrieser, C. Rusniok,A. Amend, F. Baquero, P. Berche, H. Bloecker, P. Brandt, T. Chakraborty,A. Charbit, F. Chetouani, E. Couve, A. de Daruvar, P. Dehoux, E. Domann,G. Dominguez-Bemal, E. Duchaud, L. Durant, O. Dussurget, K. D. Entian,H. Fsihi, F. G. Portillo, P. Garrido, L. Gautier, W. Goebel, N.Gomez-Lopez, T. Hain, J. Hauf, D. Jackson, L. M. Jones, U. Kaerst, J.Kreft, M. Kuhn, F. Kunst, G. Kurapkat, E. Madueno, A. Maitournam, J. M.Vicente, E. Ng, H. Nedjari, G. Nordsiek, S. Novella, B. de Pablos, J. C.Perez-Diaz, R. Purcell, B. Remmel, M. Rose, T. Schlueter, N. Simoes, A.Tierrez, J. A. Vazquez-Boland, H. Voss, J. Wehland, and P. Cossart.2001. Comparative genomics of Listenra species. Science 294:849–852,incorporated by reference herein).

Homology to bacterial conjugation proteins. The DNA sequence of 179_D14was highly homologous (88%) to the trbB gene of the phytopathogenRaistonia solanacearum, and its deduced amino acid sequence is 91%identical to the R. solanacearum probable conjugal transfer protein TrbB(Salanoubat, M., S. Genin, F. Artiguenave, J. Gouzy, S. Mangenot, M.Arlat, A. Billault, P. Brottier, J. C. Camus, L. Cattolico, M. Chandler,N. Choisne, C. Claudel-Renard, S. Cunnac, N. Demange, C. Gaspin, M.Lavie, A. Moisan, C. Robert, W. Saurin, T. Schiex, P. Siguier, P.Thebault, M. Whalen, P. Wincker, M. Levy, J. Weissenbach, and C. A.Boucher. 2002. Genome sequence of the plant pathogen Ralstoniasolanacearum. Nature 415:497–502, incorporated by reference herein).TrbB belongs to an extensive superfamily of proteins involved in theformation of surface-associated protein complexes that mediate a numberof diverse processes such as pilus biosynthesis, DNA transport, and thesecretion of virulence factors (Hobbs, M., and J. S. Mattick. 1993.Common components in the assembly of type 4 fimbriae, DNA transfersystems, filamentous phage and protein-secretion apparatus: a generalsystem for the formation of surface-associated protein complexes. Mol.Microbiol. 10:233–243; Whitchurch, C. B., M. Hobbs, S. P. Livingston, V.Krishnapillai, and J. S. Mattick. 1990. Characterization of aPseudomonas aeruginosa twitching motility gene and evidence for aspecialized protein export system widespread in eubacteria. Gene101:33–44, both of which are incorporated by reference herein). Thissuperfamily encompasses both archeal and bacterial proteins and includesthe subfamily of type IV NTPases to which TrbB proteins belong (Planet,P. J., S. C. Kachlany, R. DeSalle, and D. H. Figurski. 2001. Phylogenyof genes for secretion NTPases: identification of the widespread tadAfamily and development of a diagnostic key for gene classification.Proc. Natl. Acad. Sci USA 98:2502–2508, incorporated by referenceherein). A pentanucleotide repeat (CCGGC) in 179_D14 was also identifiedthat repeats three times and is located within the putative ORF. Thestrong DNA homology of this clone to R. solanacearum, and itsextraordinarily high G+C (68%) content compared to that of HaemophilusDNA (38%), suggests that this DNA was recently acquired by H.influenzae.

Presence of an 11-mer repeat. An 11-mer (5′-GGAATTATTTG-3′) in 162_D23was detected that repeats slightly over seven times. The H. influenzaeDNA fragment cloned in 162_D23 was only 462 bp, and the first 130 bprepresented the 3′ end of the Rd holA. Downstream of the holA homologywas a short (58 bp) region of homology (82%) to Oryza sativa genomicDNA. However, none of the DNA downstream of the probable holA hadsignificant amino acid-level homology to any known proteins. The 11-merrepeat begins approximately 180 nt downstream of holA and spans theregion of homology to O. sativa. If translated, this repeat results inthe appearance of the amino acid sequence LGIIWELFGNYLGIIWELFG in allthree positive reading frames. The slightly longer translated sequenceNYLGIIWELFGNYLGIIWFLFG appears in two of the three positive readingframes. Interestingly, the Rd genome contains one 15-mer repeat, two12-mer repeats, and a 9-mer repeating unit, none of which is repeatedmore that four times.

Codon usage of nonRd sequences. The codon usage of each of the analyzedROHs was compared to the codon usage of 71 organisms (Table 2) using aleast squares fitting procedure. Six of the 71 organisms wereHaemophilus types: three Haemophilus influenzae strains, two Haemophilusinfluenzae phage (HP 1, HP2), and Haemophilus ducreyi. It was shown (J.Gladitz et al., manuscript in preparation) that the least squaresfitting procedure used suffers an exponential loss in its power tominimize ∈ (measure of fit) with decreasing sequence lengths. In thatstudy, it was found that 50% of the analyzed H. influenzae Rd genes thatwere shorter than 160 codons had codon usage that best fit anonHaemophilus organism, yet only 10% of the H. influenzae Rd genesgreater than 0.200 codons best fit a nonHaemophilus organism.Consequently, only those nonRd ROHs listed in Table 4 that were 160codons or longer were analyzed for codon usage in this study.

A subset of 52 of the ROHs listed in Table 4 qualified for the codonstudy. The actual lengths of the sequences analyzed were, in some cases,longer than their regions of homology (ROHs) listed in Table 4; thisoccurred when the corresponding amino acid sequence continued withoutany stop codons. In some cases, the codon populations for adjacent ROHswere combined in an attempt to provide greater statistical relevance.This approach was usually applied when the protein homologies suggestedthat the ROHs originated from the same organism. In a few cases, an ROHshorter than 160 codons was combined with an adjacent ROH for analysis,regardless of their protein-level homologies; although ROHs exhibitingphage homology were not combined with ROHs without phage homology.

The organism that provided the best overall measure of fit (i.e., lowest∈-value) for each analyzed ROH is listed in Table 5. The lowest ∈-valueamongst the Haemophilus grouping is always provided, even for thosecases when the best fit was to a non Haemophilus organism. When thelowest ∈-value was to one of the two H. influenzae phage (HP1 and HP2),then the lowest ∈-value amongst the Haemophilus species was alsoincluded.

The hifC (4_E21 ROH1) and hifD (4_E21 ROH2 and 152_N2 ROH1) allelesidentified were found to have high G+C contents (45%, 46%, and 48%,respectively). The codon usage of the hifC allele (4_E21 ROH1) showedgreater similarity to the Haemophilus-specific phage HP2 than to theHaemophilus bacterial strains, though its codon usage patterns wereshown to be well-adapted to Haemophilus (Table 5). For comparison, thehifC allele from Hib strain Eagan (Watson, W. J., J. R. Gilsdorf, M. A.Tucci, K. W. McCrea, L. J. Forney, and C. F. Marrs. 1994. Identificationof a gene essential for piliation in Haemophilus influenzae type b withhomology to the pilus assembly platform genes of gram-negative bacteria.Infect. Immun. 62:468–475, incorporated by reference herein) wasanalyzed, which has a G+C content of 44%, and determined that its codonusage is also closest to that of phage HP2, with a very low ∈-value of13.45.

The hifD (4_E21 ROH2 and 152_N2 ROH1) alleles produced high ∈-values,with little similarity in codon usage to any of the sample organisms(Table 5). To determine if this atypical codon usage were unique to thehifD alleles, the hifD gene was analyzed from Hib strain Eagan for codonusage. This hifD allele has a 47% G+C content (40) and, likewise,displayed little codon-usage similarity to any sample organism. Thebest-fitting organism for the Eagan hifD was phage HP2 (∈-value=54.21).Taken together, these results indicated that H. influenzae maintains adeviant codon usage for hifD.

Unlike hifC and hifD, the fimbrial adhesin gene, hifE, does have a G+Ccontent typical of Haemophilus. Two ROHs (133_D6 and 152_N2 ROH2) wereidentified as hijE alleles, which have the highest amino-acid levelhomologies to the H. influenzae biogroup aegyptius protein (Table 4).The codon populations for these two ROHs were combined for analysis ofthese two hijE alleles since each was too short to qualify forindividual analysis (Table 4). This was justified because these ROHsrepresented different regions of the hifE locus, with no overlappingsequence. The analysis revealed H. influenzae biogroup aegyptius as thebest-fit organism (∈-value=17.74). Analysis of the type b Eagan hifEquantitatively reproduced these results, as H. influenzae biogroupaegyptius was indeed the best-fitting organism with an ∈-value of 9.15.

A hypothetical protein was identified that is highly homologous to aputative glucosidase in Yersinia pestis. This clone (43_I10) exhibitedcodon usage patterns well-adapted to Haemophilus (∈-value=14.0) and notto Yersinia (∈-value=115.45) (Table 5). The G+C content (36%) was alsoHaemophilus-like and very different from that of Yersinia (49%) (Table2). These two pieces of evidence suggest that the glucosidase inYersinia and the 43_I10 glucosidase homolog which was identified inHaemophilus shared a common ancestor and have been evolving separatelyunder the distinct selective pressures of their various host organismsfor an extended time. It appears, thus, that 43_I10 has been a longtimemember of the NTHi supra-genome as opposed to having been recentlyacquired by horizontal exchange.

The primary homologs from all three gene products in the clone 125_L2were identified as proteins encoded by a pathogenicity island fromShigella flexneri 2a. It thus appears that they may have transferredinto Haemophilus as a group, possibly from Shigella directly. When thesethree ROHs were analyzed collectively for codon usage, their resulting∈-value for S. flexneri 2a was quite poor (110.72), suggesting that theyhave been evolving separately from those genes in Shigella for anextended time. In addition, the G+C contents for all three ROHs (Table4) were very different from that of S. flexneri 2a (48%). The G+Ccontent was in fact Haemophilus-like; however, unlike the case of 43_I10where the codon usage supported the long-time presence of the sequencein Haemophilus, in this case it did not. The closest fitting organismfor this clone was enterobacteria phage T4.

Sixteen of the sequences listed in Table 5 had been shown by BLASTxanalysis to encode homologs of phage proteins. Of these, 10 displayedcodon usage patterns that revealed Haemophilus phage to be thebest-fitting organism. Two additional ROHs, 32_F13 ROH1 and 126_N4 ROH2,also have codon usage patterns best fitting that of Haemophilus phage;however, their primary BLASTx homologies were not to phage proteins.Although, in the case of 32_F13 ROH1, there were large regions ofhomology throughout the ROH to proteins from prophage CP-933C and theHaemophilus phage φ-R73.

Clones 124_K4 and 157_C17 were identified by BLASTx analysis to encodehomologs (61% identical and 66% similar, respectively) of Vibrio choleraproteins; and, V. cholerae was selected as the best-fitting organism forthese two clones (Table 5). Similarly, an excellent example of an ROHthat exhibited strong codon-usage similarity to one of thenonHaemophilus organisms in the study, while at the same time having anextremely high ∈-value for Haemophilus, is 179_D14. This clone has a 68%G+C content and likely signifies a recent acquisition by horizontaltransfer. The codon usage of this ROH was most similar to that of theG+C-rich bacterium P. aeruginosa (∈-value=19.65). The 179_D14 sequenceis 88% homologous at the nucleotide level to the trbB gene of Ralstoniasolanacearum (Table 4), a bacterium which is closely related to P.aeruginosa. P. aeruginosa, itself, has limited nucleotide homology to179_D14 (42 nt with 88% identity).

The availability of complete bacterial genomic sequences over the lastseveral years has revealed a strikingly high degree of genomic diversityamong bacteria of the same species, particularly among bacterialpathogens. Substantial differences in genome size and organization havebeen identified, in addition to a relationship between metabolicdiversity and genome size (Dobrindt, U., and J. Hacker. 2001. Wholegenome plasticity in pathogenic bacteria. Curr. Opin. Microbiol.5:550–557, incorporated by reference herein). From these studies it isbecoming apparent that bacterial pathogens share an apportionedpopulation-based genome or supra-genome, and that individual strainseach contain only a subset of the contingency genes present among theentire population (Dobrindt, U., and J. Hacker. 2001. Whole genomeplasticity in pathogenic bacteria. Curr. Opin. Microbiol. 5:550–557;Hacker, J., and E. Carniel. 2001. Ecological fitness, genomic islandsand bacterial pathogenicity: a Darwinian view of the evolution ofmicrobes. EMBO Rep. 2:376–381; Isreal, D. A., N. Salama, U. Krishna, U.M. Rieger, J. C. Atherton, S. Falkow, and R. M. Peek. 2001. Helicobacterpylori genetic diversity within the gastric niche of a single humanhost. Proc. Natl. Acad. Sci USA. 98:14625–14630, all of which areincorporated by reference herein).

It was determined among natural infecting populations of NTHi thereexists a distributed gene pool, and the extent of the NTHi supra-genomewas assessed. A highly redundant genomic library was constructed fromthe pooled DNA of ten otitis media isolates (one encapsulated and nineNTHi) and this library was used as a tool for identifying novel genesinvolved in biofilm formation and virulence. The ten clinical strainsthat comprised the pooled library underwent a minimal number ofsubcultures prior to the isolation of their genomic DNA. This ensuredthat there was essentially no decrease in selective pressure, whichcould result in the loss of particular contingency genes among thepopulations of cells in each isolated culture.

The sequence data indicate that a minimum of 9.3% of the analyzed clonesfrom the 10 clinical strains contain substantial regions of DNA notpresent in the reference strain Rd. In a smaller study, Davis et al.(Davis, J., A. L. Smith, W. R. Hughes, and M. Golomb. 2001. Evolution ofan autotransporter: domain shuffling and lateral transfer frompathogenic Haemophilus to Neisseria. J. Bacteriol. 183:4686–4635,incorporated by reference herein) obtained comparable results in whichapproximately 10% of the clones constructed from a pathogenic NTHistrain were novel relative to Rd. One finding from the sequence analysiswas that 73% of the nonRd sequences identified consisted of newlyidentified loci that have no significant homologies to any knownnucleotide sequences in any organism currently available in the publicdatabases.

The distributed genome hypothesis states that the supra-genome of agiven bacterial species includes a contingency gene pool from which eachstrain has a unique distribution when compared with all other strainscomposing the species. This is supported by the results obtained fromthe comparative analysis performed on the 10 strains used to constructthe pooled genomic library as no two strains appeared to be any morerelated to each other than to the other eight, and each strain had aunique complement of the newly identified nonRd sequences. Thesedistributed contingency genes, through reassortment during chronicinfectious processes, provide for an increased number of geneticcharacters that enable the population as-a-whole to adapt rapidly toenvironmental factors such as those experienced in the host. A corollaryto the distributed genome hypothesis is that genomic analysis of any onebacterial strain will not provide adequate information regarding thecomplete set of genes contributing to chronic pathogenicity. Geneticdiversity in the form of genomic plasticity, wherein organisms of thesame species display different genic complements (as opposed to alleliccomplements), forces a re-evaluation of the definition of a species,which now must be thought of as a population of organisms that all sharea common core set of genes with each strain or isolate having a uniquecomplement of contingency genes from a population-based supra-genome.Evidence has been accumulating from multiple groups of investigators whostudy chronic respiratory infections that multiple NTHi strains areoften carried simultaneously by the host, and that there is extensivehorizontal gene transfer among the several strains during chronicinfections. In a study by Murphy et al. (Murphy, T. F., S. Sethi, K. L.Klingrman, A. B. Brueggemann, and G. V. Doem. 1999. Simultaneousrespiratory tract colonization by multiple strains of nontypeableHaemophilus influenzae in chronic obstructive pulmonary disease:implications for antibiotic therapy. J. Infect. Dis. 180:404–409,incorporated by reference herein), it was reported that multiple strainsof NTHi were present simultaneously in the sputum of 26.3% of adultswith chronic obstructive pulmonary disease, and that these numberslikely underestimated the true frequency of the presence of multiplestrains of NTHi as they had only sampled an average of 6.3 colonies perisolate. Genomic DNA typing revealed very extensive differences in PCRband patterns among all co-infecting strains as did OMP typing bySDS-PAGE, thus arguing strongly for multiple strain infections, asopposed to strain evolution in vivo. They further determined the MICsfor 14 different antibiotics for each strain recovered, and observed inall cases very different MICs among the strains from a single clinicalisolate.

In another series of studies, Smith-Vaughan et al. (Smith-Vaughan, H.C., K. S. Sriprakash, J. D. Mathews, and D. J. Kemp. 1995. LongPCR-ribotyping of nontypeable Haemophilus influenzae. J Clin Microbiol.33:1192–1195; Smith-Vaughan, H. C., A. J. Leach, T. M. Shelby-James, K.Kemp, D. J. Kemp, and J. D. Mathews. 1996. Carriage of multipleribotypes of non-encapsulated Haemophilus influenzae in Aboriginalinfants with otitis media. Epidemiol. Infect. 116:177–183;Smith-Vaughan, H. C., K. S. Sriprakash, J. D. Mathews, and D. J. Kemp.1997. Nonencapsulated Haemophilus influenzae in Aboriginal infants withotitis media: prolonged carriage of P2 porin variants and evidence forhorizontal P2 gene transfer. Infect Immun. 65:1468–1474, all of whichare incorporated by reference herein) described simultaneous carriageand horizontal gene transfer among multiple NTHi strains in AustralianAboriginals. In these studies they document numerous cases of horizontaltransfer (among individual strains within infectious isolates) of thegene encoding the major outer membrane protein P2. These investigatorsstate that the findings of identical P2 sequences in different geneticbackgrounds, and dissimiliar P2 sequences in similar geneticbackgrounds, suggest that horizontal gene transfers make a significantcontribution to the diversity of H. influenzae. They further note thatthe evidence for horizontal gene transfer indicates that genes otherthan P2 are also subject to immune selection as otherwise there would belittle advantage for a P2 variant in moving from one genetic backgroundto another. They note that their analysis of H. influenzae strains fromsingle chronically infected persons present a complex picture ofmultiple insertions, deletions and substitutions of amino acids, andthat horizontal transmission of P2 gene segments provides the mostlikely mechanisms for genetic diversification. Moreover, they note thatchronic infections which are associated with the carriage of largenumbers of bacteria distributed among multiple strains would provide anenvironment that favored horizontal gene transfer, and the resultantreassortment would lead to long-term persistence of H. influenzae due tosequential carriage of recombinants with improved fitness. Finally, theystate that their data favor P2 evolution by selection and provide noevidence of antigenic drift among sequential isolates. They concludedthat horizontal gene transfer in H. influenzae was expected as theseorganisms are naturally competent, and that co-colonization withmultiple types at the same time would provide the opportunity for genetransfer (Smith-Vaughan, H. C., K. S. Sriprakash, J. D. Mathews, and D.J. Kemp. 1997. Nonencapsulated Haemophilus influenzae in Aboriginalinfants with otitis media: prolonged carriage of P2 porin variants andevidence for horizontal P2 gene transfer. Infect Immun. 65:1468–1474,incorporated by reference herein).

It is true that the above reports on NTHi evolution confine themselveslargely to single gene systems, particularly those known to be involvedwith surface proteins. However, they do not favor the hypothesis thatgenetic variants have arisen primarily by point mutations, nor is thereany reason to suppose a priori that similar horizontal gene transfermechanisms are not operative over the entire H. influenzae genome asnearly all H. influenzae genes, including many of the novel sequencesreported here, have USSs to aid in uptake, retention and transformation.Moreover, Ochman and Lawrence (Ochman, H., J. G. Lawrence, and E. A.Groisman. 2000. Lateral gene transfer and the nature of bacterialinnovation. Nature 405:299–304, incorporated by reference herein) foundthat bacterial evolution occurs largely through horizontal genetransfer, and that this mechanism produces extremely dynamic genomes inwhich substantial amounts of DNA are introduced into and deleted fromthe chromosome. Lawrence (Lawrence, J. 2001. Catalyzing bacterialspeciation: correlating lateral transfer with genetic headroom. SystBiol. 50:479–496, incorporated by reference herein) further showed thatthe amounts of foreign DNA in bacterial genomes, and the rate at whichthis DNA is acquired, are consistent with gene transfer as the primarycatalyst for microbial differentiation.

The finding that many natural NTHi infections are polyclonal, and thatstrains evolve in vivo (Murphy, T. F., S. Sethi, K. L. Klingman, A. B.Brueggemann, and G. V. Doem. 1999. Simultaneous respiratory tractcolonization by multiple strains of nontypeable Haemophilus influenzaein chronic obstructive pulmonary disease: implications for antibiotictherapy. J. Infect. Dis. 180:404–409; van Alphen, L., D. A. Caugant, B.Duim, M. O'Rourke, and L. D. Bowler. 1997. Differences in geneticdiversity of nonencapsulated Haemophilus influenzae from variousdiseases. Microbiology 143:1423–1431, both of which are incorporated byreference herein), suggests that during chronic infections thehorizontal reassortment of genes from the infecting-populationsupra-genome may act as a supra-virulence factor. A supra-virulencefactor is defined as a trait which exists at the population level, asopposed to the individual bacterial cell level, that provides forincreased bacterial survival or pathogenicity at the expense of thehost. The extensive genomic-plasticity results reported herein supportthe concept that polyclonal NTHi infections would provide sufficientgenomic fodder to allow for nearly limitless numbers of recombinants,both allelic and genic. Thus, with the generation of large numbers ofrecombinant bacteria, the probability would be greatly increased, whencompared with a clonal infection, of a strain appearing that displayed asignificant survival advantage for the environmental conditionsencountered in a particular host. In fact, the use of clonal isolatesfor in vivo infection models may, in part, explain why it is oftendifficult to recapitulate chronic or persistent infections inexperimental animals.

The data presented here supports the concept that the NTHi possess apopulation-based supra-genome and that no two strains have the samecomplement of genes. Moreover, it would appear as if the NTHIsupra-genome is necessarily much larger in size than the genomes ofindividual bacteria.

The distributed genome hypothesis correlates well with the finding thatchronic bacterial infections of the middle-ear and other organs areoften associated with biofilm formation (Ehrlich, G. D., R. Veeh, X.Xang, J. W. Costerton, J. D. Hayes, F. Z. Hu, B. J. Daigle, M. D.Ehrlich, and J. C. Post. 2002. Mucosal biofilm formation in middle-earmucosa in the chinchilla model of otitis media. JAMA 287:1710–1715;Post, J. C. 2001. Direct evidence of bacterial biofilms in otitis media.Laryngoscope 111:2083–2094; Post, J. C., and G. D. Ehrlich. 2000. Theimpact of the polymerase chain reaction in clinical medicine. JAMA283:1544–1546; Rayner, M. G., Y. Zhang, M. C. Gorry, Y. Chen, J. C.Post, and G. D. Ehrlich. 1998. Evidence of bacterial metabolic activityin culture-negative otitis media with effusion. JAMA 279:296–299, all ofwhich are incorporated by reference herein), as biofilms are known toprovide a fertile environment for horizontal gene transfer (Hausner, M.,and S. Wuertz. 1999. High rates of conjugation in bacterial biofilms asdetermined by quantitative in situ analysis. Appl. Environ. Microbiol.65:3710–3713, incorporated by reference herein). Moreover, it hasrecently been suggested that DNA is a major component of theextracellular biofilm matrix of the gram-negative opportunistic pathogenPseudomonas aeruginosa (Whitchurch, C. B., T. Tolker-Nielsen, P. C.Ragas, and J. S. Mattick. 2002. Extracellular DNA required for bacterialbiofilm formation. Science 295:1487, incorporated by reference herein).An understanding of the extent of genomic plasticity in bacterialpathogens such as NTHi and P. aeruginosa should provide insight intoclinically relevant topics such as biofilm development and expression ofvirulence factors.

Claverys et al. (Claverys, J. P., M. Prudhomme, I. Mortier-Barriere, andB. Martin. 2000. Adaptation to the environment: Streptococcuspneumoniae, a paradigm for recombination-mediated genetic plasticity?Mol Microbiol. 35:251–259, incorporated by reference herein) haveindependently put forth a theorem of bacterial diversity from theirstudies of the gram-positive Streptococcus pneumoniae that incorporatessome of the same emergent concepts developed hereby studying thegram-negative pathogens H. influenzae (vidae supra) and Pseudomonasaeruginosa (data not shown). In their model, substitutive recombinationamong DNAs from other pneumococci is the most common form of geneacquisition. However, they also recognize diversity generation throughinsertion of capsular genes and the creation of mosaic genes throughiterative homologous recombination events. These investigators statethat, through transformation, each pneumococcus has access to a ‘global’genome that is larger than the genome of any single bacterium. They goon to estimate that the global genome may be approximately 0.5 Mb largerthan a single bacterium's. Thus, they understand the issue, but withoutthe large-scale genomic comparative data contained in this report, theywere not able to fully appreciate the extent of genomic plasticity northe size and scope of the supra-genomic reservoir.

It is maintained that the degree of plasticity among strains of H.influenzae is considerably greater than what is reported. More stringentrequirements were imposed on the sequences that were classified as novelthan for those classified as Rd. Seemingly novel clones were knowinglyexcluded either because their internal sequences could not be obtainedeasily or because the level of ambiguity in their sequences was >5% overthe length of the cloned fragment. Moreover, isolates were analyzed thatwere collected from one infection site, the middle ear, and in onegeographical location. An even higher percentage of novel DNA sequenceslikely would be detected among strains acquired from a variety ofinfection sites and from healthy carriers. Indeed, van Alphen et al.(van Alphen, L., D. A. Caugant, B. Duim, M. O'Rourke, and L. D. Bowler.1997. Differences in genetic diversity of nonencapsulated Haemophilusinfluenzae from various diseases. Microbiology 143:1423–1431,incorporated by reference herein) reported that the genetic diversity ofNTHi strains taken from patients with otitis media is less than thatseen in strains isolated from healthy carriers or from patients withchronic disease.

A number of sequences already known to contribute to H. influenzaepathogenesis emerged in the set of contingency genes, including thoserequired for tryptophan catabolism and for the biosynthesis of variousadhesins. Also, proteins from phage and restriction/modification (R/M)systems constitute 22% of the homologs listed in Table 4, as phage areoften associated with horizontal gene transfer (Dobrindt, U., and J.Hacker. 2001. Whole genome plasticity in pathogenic bacteria. Curr.Opin. Microbiol. 5:550–557; Hacker, J., G. Blum-Oehler, I. Mühldorfer,and H. Tschäpe. 1997. Pathogenicity islands of virulent bacteria:structure, function and impact on microbial evolution. Mol. Microbiol.23:1089–1097, both of which are incorporated by reference herein), andR/M gene clusters are emerging as part of the flexible gene pool of aprokaryotic genome (Hacker, J., and E. Carniel. 2001. Ecologicalfitness, genomic islands and bacterial pathogenicity: a Darwinian viewof the evolution of microbes. EMBO Rep. 2:376–381; Kobayashi, I., A.Nobusato, N. Kobayashi-Takahashi, and I. Uchiyama. 1999. Shaping thegenome: restriction-modification systems as mobile genetic elements.Curr. Opin. Genet. Dev. 9:649–656, both of which are incorporated byreference herein). Yet the majority of the homologies displayed by theputative products of the nonRd sequences Were to a wide array ofproteins found not only in bacteria, but also in protozoans, metazoans,viruses and fungi.

Also as part of the support for a Haemophilus supra-genome theory, codonusage was used to determine whether the nonRd DNA sequences evidencedpatterns typical of Haemophilus. These unique sequences, assuming amaximum parsimony model, can be attributed to at least three subsets.The first are those that are Haemophiloid in nature, meaning that boththeir G+C content and their codon usage are most similar to Haemophiluswhen compared against 71 diverse prokaryotic and eukaryotic genomes.This suggests that many of these newly identified contingency genes havebeen evolving within the Haemophilus supra-genome for very extendedperiods of time. Often this was the case even when the nucleotide-and/or protein-level homologies strongly implied that the sequence wassimilar to one in a distantly related organism. Of course, a number ofsequences in this category may have been acquired more recently throughhorizontal transfer from a closely related bacterium. The second subsetof unique sequences are those that display a G+C content similar toHaemophilus, but a codon usage not fitting the general Haemophiluspattern. These sequences have likely been in the Haemophilussupra-genome for an intermediate period of time and likely are examplesof DNA in a state of codon flux following horizontal transfer. The thirdsubset of unique sequences most likely represent additions to theHaemophilus supra-genome from distantly related organisms, as theydiffer both in terms of G+C content and codon usage. The hifD allele isfound in this category, which may suggest that the hif pathogenicityisland is relatively new to the H. influenzae supra-genome. Of course,there may be specific situations in which an uncharacteristic codonusage is acceptable or even advantageous to the organism (Hacker, J.,and J. B. Kaper. 1999. The concept of pathogenicity islands, p. 1–11. InJ. B. Kaper, and J. Hacker (ed.), Pathogenicity islands and other mobilevirulence elements. American Society for Microbiology, Washington, D.C.;Moszer, I., E. P. C. Rocha, and A. Danchin. 1999. Codon usage andlateral gene transfer in Bacillus subtilis. Curr. Opin. Microbiol.2:524–528, both of which are incorporated by reference herein). Overall,these subsets strongly indicate that Haemophilus is constantly acquiringnew DNA sequences and that the contingency pool is, indeed, dynamic.

Although the invention has been described in detail in the foregoingembodiments for the purpose of illustration, it is to be understood thatsuch detail is solely for that purpose and that variations can be madetherein by those skilled in the art without departing from the spiritand scope of the invention except as it may be described by thefollowing claims.

1. An isolated DNA sequence of Haemophilus influenzae clone 125_L2 shownin SEQ. ID. NO. 2.